Fragmented Lactic Acid Bacterial Cells Activate Peroxisome

Feb 29, 2016 - To identify an effective PPAR agonist, we screened the in vitro PPARα/γ activation ability ... PPARα and PPARγ derived from LAB cel...
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Fragmented lactic acid bacterial cells activate peroxisome proliferatoractivated receptors and ameliorate dyslipidemia in obese mice Futoshi Nakamura, Yu Ishida, Daisuke Sawada, Nobuhisa Ashida, Tomonori Sugawara, Minami Sakai, Tsuyoshi Goto, Kawada Teruo, and Shigeru Fujiwara J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05827 • Publication Date (Web): 29 Feb 2016 Downloaded from http://pubs.acs.org on March 14, 2016

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Journal of Agricultural and Food Chemistry

Fragmented lactic acid bacterial cells activate peroxisome proliferator-activated receptors and ameliorate dyslipidemia in obese mice

Futoshi Nakamura,† Yu Ishida,† Daisuke Sawada,† Nobuhisa Ashida,† Tomonori Sugawara,† Manami Sakai,‡ Tsuyoshi Goto,‡ Teruo Kawada,‡ and Shigeru Fujiwara*†



Research & Development Center, Asahi Group Holdings, Ltd., 5–11–10 Fuchinobe,

Chuo-ku, Sagamihara-shi, Kanagawa 252-0206, Japan ‡

Laboratory of Molecular Function of Food Division of Food Science and

Biotechnology, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan

*

Corresponding author: [email protected]

Phone: +81-42-769-7828; Fax: +81-42-769-7810.

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Abstract

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Recent studies suggest that peroxisome proliferator-activated receptor (PPAR)

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activation ameliorates metabolic disorders, including dyslipidemia. To identify an

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effective PPAR agonist, we screened the in vitro PPARα/γ activation ability of organic

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solvent extracts from food-oriented bacterial strains belonging to five genera and 32

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species, including lactic acid bacteria, and of these, Lactobacillus amylovorus CP1563

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(CP1563) demonstrated the highest PPARα/γ agonist activity. We also found that

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physical fragmentation of the strain could substitute organic solvent extraction for the

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expression of CP1563 activity in vitro. For functional food manufacturing, we selected

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the fragmented CP1563 and conducted subsequent animal experiments. In an obese

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mouse model, we found that treatment with fragmented CP1563 for 12 weeks decreased

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the levels of LDL-cholesterol and triglyceride in plasma, significantly decreased the

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atherosclerosis index and increased the plasma HDL-cholesterol level. Thus, we

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conclude that fragmented CP1563 may be a candidate for the prevention and treatment

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of dyslipidemia.

16 17

Key words

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lactic acid bacteria, cell fragmentation, peroxisome proliferator-activated receptors,

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dyslipidemia

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Introduction

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Metabolic syndrome is a risk factor for chronic diseases, such as cardiovascular disease,

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hypertension and diabetes.1,2 Dyslipidemia is a direct risk factor for atherosclerotic

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cardiovascular disease and is characterized by a high level of triglycerides (TGs) and a

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low level of high-density lipoprotein (HDL) cholesterol.3,4 Low-density lipoprotein

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(LDL) cholesterol is also a well-known and strong cardiovascular risk factor. Thus,

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maintaining the optimal levels of these factors is a critical issue.

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Previous studies have reported the plasma cholesterol-ameliorating effects of

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fermented milk,5-8 and animal studies have shown that lactic acid bacteria (LAB) exert

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anti-dyslipidemic effects, including decreases in the serum and liver cholesterol

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concentrations.9 The mechanisms through which LAB improve lipid metabolism include

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cholesterol and/or bile acid adsorption, bile acid deconjugation and pancreatic lipase

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inhibition in the gut.10 However, other studies have suggested that the oral

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administration of fermented milk has a negligible effect on lipid metabolism,11-15 and

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the cholesterol-lowering function and working mechanism16 of LAB remain

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controversial.

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Our interest focused on peroxisome proliferator-activated receptor (PPAR)

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agonists, namely PPAR agonist(s) for PPARα and PPARγ derived from LAB cells and

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their metabolites. PPARs, which are members of the nuclear receptor superfamily, are

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ligand-activated transcription factors.17 PPARα is expressed primarily in the liver and

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skeletal muscle and promotes fatty acid β-oxidation to reduce the amount of TGs in the

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blood, liver and skeletal muscle.18 By acting as PPARα agonists, fibrate

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anti-hyperlipidemic drugs down-regulate plasma TGs and up-regulate HDL-cholesterol.

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In contrast, PPARγ is highly expressed in adipose tissue and is indispensable for the

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regulation of metabolic processes such as adipocyte differentiation, lipid storage and

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glucose metabolism.19 Thiazolidinedione, which is a PPARγ agonist, has been approved

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for the treatment of insulin resistance. Moreover, pan-PPAR agonists and dual-PPAR

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agonists are expected to effectively treat metabolic disorders, including

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dyslipidemia.20,21 For example, PPARα/γ and PPARα/δ agonists, such as glitazars and

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GFT505, are important candidates for treating diabetes. Saroglitazar, a PPARα/γ agonist,

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has been approved for the treatment of type II diabetes.22

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We hypothesized that LAB function as PPARα/γ agonists and promote the control

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of dyslipidemia and lifestyle-related diseases. In this study, we compared the

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PPAR-activation abilities of diethyl ether extracts of various LAB strains and food

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microorganisms in vitro and found that the LAB strain CP1563 possesses potent

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PPARα/γ dual agonist activity. We hypothesized that unique fatty acids, such as

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13-oxo-9,11-octadecadienoic acid23 and/or other hydrophobic materials, including

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glycerin esters in the CP1563 cell membrane and cytoplasm, could function as PPARα/γ

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ligands and confirmed that CP1563 after the fragmentation process can efficiently

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release the PPARα/γ activator(s). The finding that CP1563 functions after fragmentation

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is attractive because of the possible advantages of using CP1563 in industrial

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applications24 as a “paraprobiotic”25 for metabolic syndrome. In this study, we

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attempted to investigate the possibility of using fragmented CP1563 cells as a

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paraprobiotic for improving dyslipidemia in mice fed a high-fat diet.

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Materials and Methods

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Bacterial Culture

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Various LAB strains and food microorganisms (Table 1), which were stocked in our

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culture collection library, were cultured. Strains of Enterococcus, Issatchenkia,

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Lactobacillus, Lactococcus and Leuconostoc were cultured in MRS broth (Becton

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Dickinson and Company, Sparks, MD, USA) at 37 °C under a partially anaerobic

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atmosphere. Bifidobacteria were cultured in Briggs liver broth at 37 °C under a strict

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anaerobic atmosphere, and potato dextrose broth was used to cultivate yeast at 30 °C

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and 25 °C under aerobic conditions. The culture duration was 18 h in all cases. The

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resulting bacteria or yeast cells were harvested by centrifugation (18,300 x g and 4 °C

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for 10 min) (Hitachi Himac CR21GII, Hitachi, Ltd., Tokyo, Japan), washed successively

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with MilliQ water and lyophilized.

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Luciferase Assays for PPAR Ligand Activity

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To saponify the bacterial cells, the dried cells were suspended in 500 ml of 0.5 M

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ethanolic potassium hydroxide solution (Cat. No. 32816-08, Kanto Chemical Industry

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Co., Ltd., Tokyo, Japan) per 5 g of bacteria, and the solutions were sonicated at 300 W

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for 2 min (VC-750, Tokyo Rikakikai Co., Ltd., Tokyo, Japan). The bacterial

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preparations were then boiled for 1 h. After the solutions were cooled within a jet of

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water, their pH values were adjusted to 2 or less with concentrated hydrochloric acid

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(Wako Pure Chemical Industries, Ltd., Osaka, Japan) to release the fatty acids from the

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bacterial cells. The mixtures were reduced to 50-ml volumes using a rotary evaporator

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in a water bath at 40 °C, and an equal volume of diethyl ether was added (Wako Pure

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Chemical Industries, Ltd., Osaka, Japan). The samples were then agitated for 1 h using a

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shaker (SH-100, KURABO Industries, Ltd., Tokyo, Japan). The upper layer was

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extracted four times with diethyl ether, and the combined diethyl ether extracts were

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dried by evaporation. Ether extracts from the bacteria were diluted with a

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commercialized medium for animal cell culture, Opti-MEM (Cat. No. 31985-062,

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Thermo Fisher Scientific, Inc., Yokohama, Japan). The fatty acid concentration was

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determined using the Free Fatty Acid Quantification Kit provided by Wako Pure

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Chemical Industries, Ltd. (Osaka, Japan) and adjusted to a final concentration of 2.5 µM,

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which was calculated based on fatty acid equivalents.

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The CP1563 bacteria were passaged three times in medium consisting of

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food-grade ingredients. The cells were harvested by centrifugation, washed with MilliQ

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water, and killed by heating at 90 °C. The bacteria were disrupted using a DYNO-MILL

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(Willy A. Bachofen, Muttenz, Switzerland). The fragmented samples were lyophilized

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for the subsequent animal studies and then analyzed using a scanning electron

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microscope (VHX-D500, KEYENCE, Osaka, Japan) and the WinROOF image

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processing software (Mitani Corporation, Fukui, Japan).

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A luciferase assay using the GAL4/PPAR chimera system was employed to assess

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the agonistic activation of PPARα/γ according to previously published standards.26,27 In

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brief, monkey kidney CV1 cells were cultured in Dulbecco’s modified Eagle’s medium

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(DMEM) (Sigma-Aldrich Japan, Tokyo, Japan) supplemented with 10% fetal bovine

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serum (FBS 172012-500ML; Sigma-Aldrich Japan, Tokyo, Japan) at 37 °C in a

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humidified 5% CO2 atmosphere. After 24 h, one of three plasmids, namely

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p4xUASg-tk-luc (a reporter plasmid), pM-hPPARα or pM-hPPARγ (an expression

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plasmid for a chimera protein consisting of the GAL4 DNA-binding domain and human

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PPARα- or PPARγ-ligand binding domain) and pRL-CMV (an internal control for

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normalizing the transfection efficiencies), were transfected into CV1 cells in a

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24-well-plate format using Lipofectamine Plus (Invitrogen Japan, Tokyo, Japan). After

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transfection, the cells were cultured for 24 h, and the compounds for the ligand assays

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were added to the medium at the appropriate concentrations using the serial dilution

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method. The cells were then incubated for 24 h, lysed and subjected to a luciferase

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assay using the Dual-GloTM Luciferase Reporter Gene Assay system (Promega KK,

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Tokyo, Japan). The assays were performed using diethyl ether extracts obtained from

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various LAB strains, food microorganisms and GW7647 (at a final concentration of 10

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nM, Sigma-Aldrich Japan, Tokyo, Japan) as a positive control for PPARα activation.

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Troglitazone (at a final concentration of 1 µM; Wako Pure Chemical Industries, Ltd.,

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Osaka, Japan) was used as a positive control for PPARγ ligand activation.

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First, the PPARα agonist activity in extracts derived from 38 bacterial strains was

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evaluated, and the PPARα agonist activity in seven L. amylovorus strains was then

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assessed. CP1563 extracts were also compared with known PPARα ligands, namely

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(-)-epigallocatechin gallate (EGCG; at a final concentration of 75, 100, 150 or 200 µM,

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ChromaDex, Inc., CA, USA), eicosapentaenoic acid (EPA; at a final concentration of

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0.625, 1.25, 2.5 or 5 µM, Nippon Suisan Kaisha, Ltd., Tokyo, Japan) and

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9(Z),11(E)-octadecadienoic acid (conjugated linoleic acid, CLA; at a final concentration

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of 2.5 µM, Matreya, LLC, PA, USA).

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Animal Experiments

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Male C57BL/6N mice (5 weeks of age) were obtained from Charles River Laboratories

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Japan, Inc. (Kanagawa, Japan). The mice were housed in an air-conditioned room that

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was maintained at 23 ± 1.5 °C with a relative humidity of 55 ± 15% under a constant

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12-h light/12-h dark cycle (with light from 8:00 a.m. to 8:00 p.m.). Table 2 shows the

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compositions of the experimental diets. For all experiments, the dietary components

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were mixed and used to produce pelleted feed. The CP1563 cells were passaged three

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times in a medium containing all food-grade ingredients. Lyophilized bacterial cell

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preparations were processed using a food processor mill (TESCOM, Tokyo, Japan) to

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remove any clots and then mixed with CE-2 animal chow powder (CLEA Japan, Inc.,

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Tokyo, Japan). The pelleted diet was produced by Oriental Yeast Co., Ltd. (Tokyo,

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Japan). The pelleted diet with 1% CP1563 contained the equivalent of approximately

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5.0 × 1011 cells per gram.

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To acclimate the mice to the experimental conditions, the animals were housed in

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rooms for six to eight days prior to the experiments and were fed a normal diet (CE-2,

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CLEA Japan, Inc., Tokyo, Japan) or a high-fat (HF) control diet.28,29 The mice were

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divided into several groups and evaluated using a randomized block design. The mice

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were pair-fed over a series of experiments and sacrificed after one day of fasting to

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collect whole-body blood samples and measure their visceral fat.

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This research was approved by the research ethics committee of the R&D Center

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at Asahi Group Holdings, Ltd. (permit numbers: 07-014, 08-007, 08-020 and 09-006).

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The animals were treated in accordance with the ethical guidelines for animal care,

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handling and sacrifice of the R&D Center at Asahi Group Holdings, Ltd. All surgeries

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were performed under inhalation anesthesia with isoflurane (Escain; Mylan Seiyaku,

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Osaka, Japan), and all efforts were made to minimize suffering.

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Preliminary Animal Experiments

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In preliminary experiment 1, the mice were fed a normal diet for six days and then

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housed with free access to a HF control diet for five days. The mice were then divided

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into four groups (n = 24 per group) as follows: 1) a HF diet-fed control group, 2) a 1%

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fragmented CP1563-containing diet-fed group, 3) a 0.05% fenofibrate-containing

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diet-fed group and 4) a 0.5% fenofibrate-containing diet-fed group. The mice were fed

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their assigned diet for two weeks and then sacrificed under anesthesia with isoflurane

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for animals (Mylan Seiyaku, Osaka, Japan). The liver weights and visceral fat around

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the epididymides were measured upon dissection. In preliminary experiment 2, the mice

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were acclimated to their cages for seven days and were then housed with free access to

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the HF control diet for six days. The mice were then divided into three groups (n = 24

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per group) as follows: 1) a HF diet-fed control group, 2) a 1% fragmented

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CP1563-containing diet-fed group and 3) a 0.05% fenofibrate-containing diet-fed group.

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The mice were fed their assigned diets for six weeks and were then sacrificed. The liver

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weights and visceral fat around the epididymides, kidneys and gut were measured upon

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dissection.

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Comparative Animal Experiment between Intact CP1563 and the Fragmented

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Bacterial Cells Prepared with a Size Reduction Mill

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The mice were housed in animal rooms for eight days with free access to the HF control

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diet and tap water. The mouse body weights, food consumption and visceral fat volumes

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were measured using a CT scanner (Latheta LCT-100, Hitachi-Aloka Medical, Ltd.,

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Tokyo, Japan), and the mice with body weights within the mean ± 1.5 S.D. were then

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selected and randomly divided into three groups (n = 30 per group) as follows: 1) a HF

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diet-fed control group, 2) a 1% intact CP1563-containing diet-fed group and 3) a 1%

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fragmented CP1563-containing diet-fed group. The mice were fed their assigned diets

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for six weeks, and the visceral fat volumes after six weeks were measured using a CT

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scanner.

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Long-term Administration of Fragmented CP1563

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The mice were acclimated and then selected under the same conditions as in the

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previous comparative experiment. The mice were divided into two groups (n = 50 per

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group): a HF diet-fed control group and a 1% fragmented CP1563-containing diet-fed

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group. The mice were fed for 12 weeks and then sacrificed, and the visceral fat volumes

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were measured using a CT scanner.

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Dose Dependency of Fragmented CP1563 in Animal Studies

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The mice were acclimated and selected using the same method as previously described.

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The mice were divided into four groups (n = 36 per group) as follows: 1) a HF diet

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control group, 2) a 0.25% fragmented CP1563-containing diet group, 3) a 0.5%

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fragmented CP1563-containing diet group and 4) a 1% fragmented CP1563-containing

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diet group. The mice were fed their assigned diets for 6 weeks and then were sacrificed.

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The visceral fat volumes were measured using a CT scanner.

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Correlation between Degree of Fragmentation of CP1563 and PPARα Ligand Activity

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in vitro

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Eighteen fragmented CP1563 preparations were produced by combinations of different

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mills (six different jet mills, two different planetary mills, and DYNO-MILL) and

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different fracturing conditions. Three types of dry-type jet mills (FS-4, CO-JET and

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STJ-200, Seishin Enterprise Co., Ltd., Tokyo, Japan) were simultaneously run four

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times in turn. We obtained 12 different preparations with different long dimensions from

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these three dry jet mills using four different running conditions. Three other types of

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dry-type jet mills [SK-Jet-O-Mill and FS-4 (vertical type), Seishin Enterprise Co., Ltd.,

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Tokyo, Japan; Jet-O-Mizer, Sansho Industry Co., Ltd., Osaka, Japan] were run once,

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and we obtained three other types of fragmented bacterial preparations with different

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long dimensions. Furthermore, two types of planetary mills (SKF-04, Seishin Enterprise

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Co., Ltd., Tokyo, Japan; GOT5, Sansho Industry Co., Ltd., Osaka, Japan) and a

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DYNO-MILL (Multi-lab, Shinmaru Enterprises Corporation, Osaka, Japan) were used

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to disrupt the bacterial cells, and we thus obtained three other types of fragmented

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bacterial cell preparations with different long dimensions. PPARα reporter assays were

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performed as described previously with a concentration of 625 µg of each of the

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fragmented bacterial preparation/mL in 24-well culture plates. These assays were

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conducted with the intact cell preparation and 18 fragmented bacterial cell preparations

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of CP1563.

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Correlations between the Degree of Fragmentation of CP1563 and PPARα Ligand

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Activity in vitro and HDL-Cholesterol Level-Raising Activity in vivo

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Three different preparations of fragmented CP1563 were prepared using DYNO-MILL

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(Multi-lab, Shinmaru Enterprises Corporation, Osaka, Japan) and two other crushing

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machines (Jet-Mill: Jet-O-Mizer, Sansho Industry Co., Ltd., Osaka, Japan and Planetary

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Mill: GOT5, Sansho Industry Co., Ltd., Osaka, Japan). The long dimensions of these

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preparations were measured, and the preparations (at a final concentration of 625

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µg/mL) and GW7647 as a positive control were applied to the PPARα reporter assay.

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These preparations were added to the basal diet at a 1% level as described previously,

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and the resulting diets were used for an animal experiment aiming to detect the plasma

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HDL-cholesterol-raising activity in mice fed these diets for six weeks in a manner

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similar to that described above.

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Measurement of Biological Markers in Plasma Samples

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The total cholesterol and TG levels in the plasma were determined using a Fuji

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DRI-CHEM X (FUJIFILM Corporation, Tokyo, Japan), and the HDL-cholesterol level

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was measured using the HDL-Cholesterol E-test (Wako Pure Chemical Industries, Ltd.,

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Osaka, Japan). The level of LDL-cholesterol was estimated using the Friedewald

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formula.30 The atherosclerosis index (AI) was calculated as follows: AI = (total

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cholesterol – HDL-cholesterol) ÷ HDL-cholesterol. The level of high-molecular-weight

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adiponectin was measured using an enzyme-linked immunosorbent assay (ELISA) kit

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(Shibayagi Co., Ltd., Gunma, Japan).

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Statistical Analyses

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All of the statistical analyses were performed using SPSS ver. 20 software (IBM Japan.,

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Tokyo, Japan). The results from all experiments are expressed as the means ± standard

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deviations (S.D.). A two-way analysis of variance was applied, and post-hoc Dunnett’s

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multiple-comparison procedure was then used to compare the averages of several

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measured items between the groups. The differences were considered significant if the

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p-value was less than 0.05.

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Results

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The Organic CP1563 Extract Showed High PPARα/γ Activation Activity

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We evaluated the ability of 25 bacterial strains from our culture stocks and 20 recently

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isolated strains of bacteria and yeast to activate PPARα signaling (Table 1). Seven LAB

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strains could not be harvested from the growth media for the preparation of ether

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extracts. As shown in Figure 1A, many bacterial strains exhibit PPARα agonist activity

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in their diethyl ether extracts, and the relative activities of 17 of the tested strains

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exceeded 50% of that of the positive control. In particular, L. amylovorus CP1563

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showed the highest agonist activity, with a 1.5-fold higher strength than that of the

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GW7647-positive control. Eight LAB strains that showed high PPARα agonist activity

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were then analyzed to determine the ability of their ether extracts to activate PPARγ

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(Fig. 1B). We examined the PPARγ activation activities of eight strain extracts and

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found that five of the eight extracts demonstrated PPARγ activation activity. We

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examined the PPARα agonist activity of seven L. amylovorus strains, including the type

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strain (ATCC 33620T), and found that CP1563 showed the highest activation ability (Fig.

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2). As shown in Figure 3, the agonist activity of CP1563 was higher than that of the

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food-oriented PPARα ligands (i.e., EGCG, EPA and CLA) under our experimental

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conditions. At approximately the same molecular concentration, the activity of the crude

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organic CP1563 extract showed two-fold higher PPARα activity compared with CLA

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and EPA. In addition, the PPARα activation ability of the CP1563 extract (2.5 µM: fatty

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acid equivalent) was higher than those of the higher concentrations of EPA (5 µM) and

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EGCG (200 µM). Based on the results of these reporter assays, we selected CP1563 for

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subsequent experiments to determine the PPAR ligand(s) in the extracts and fragmented

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bacterial cells, which may be a robust source of potent PPAR ligand(s) and could be

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useful as a food-processing ingredient.

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The Plasma HDL-cholesterol Concentration Increased in Fragmented CP1563-fed

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Mice

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Preliminary experiments were performed to determine the effect of CP1563 on

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dyslipidemia and to refine the experimental conditions in vivo. Figures 4 and 5 show the

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PPARα agonist activities in mice fed a diet containing 1% fragmented CP1563

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(approximately 1.5 × 1010 cells/day) for two and six weeks, respectively. Feeding

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animals a 0.5% fenofibrate-containing diet increased the HDL-cholesterol level and

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tended to decrease the LDL-cholesterol level. A two-week feeding period of fragmented

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CP1563 (Fig. 4) did not significantly increase the plasma HDL-cholesterol level. In

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contrast, a six-week feeding period (Fig. 5) increased the plasma HDL-cholesterol

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increased and significantly increased the AI in the positive control and the fragmented

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CP1563-fed groups (p < 0.001). The liver weights were significantly increased in the

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positive control group and in the group fed fragmented CP1563 for six weeks (Table 3).

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However, no significant changes in the visceral fat weights were noted in either the two-

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or six-week feeding experiments (Table 3).

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Fragmentation of LAB and Food Microorganisms is Required to Observe an Effect

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on Dyslipidemia

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We compared the effects of fragmented CP1563 with unprocessed CP1563 in the

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diet-induced obese mouse model to assess the influence of fragmentation on the effects

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of CP1563 in vivo. The electron micrographs of the unprocessed (Fig. 6A) and fully

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fragmented CP1563 (Fig. 6B) show that CP1563 was pulverized into small pieces by

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the mechanical fragmentation process. In the 1% intact CP1563-fed group

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(approximately 1.5 × 1010 cells/day), no significant change in any plasma biomarker was

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detected (Fig. 7). In contrast, the 1% fragmented CP1563-fed group (approximately 1.5

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× 1010 cells/day) presented a significant increase in the plasma HDL-cholesterol

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concentration (p < 0.001) and a significantly decrease in the resulting AI (p < 0.05).

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However, no significant difference in the visceral fat weights was observed in either

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group (Table 4).

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Long-term Administration of Fragmented CP1563 Resulted in Anti-dyslipidemic

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Effects

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The administration of fragmented CP1563 cells (approximately 1.5 × 1010 cells/day) for

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12 weeks resulted in an elevation in the plasma HDL-cholesterol level (p < 0.001) and

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decreases in the plasma LDL-cholesterol (p < 0.05) and plasma TG levels (p < 0.05) and

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the AI (p < 0.001; Figs. 9A-9D). The concentration of high-molecular-weight plasma

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adiponectin (Fig. 9E), liver weight and visceral fat weight were found to be regulated

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(Table 5), but these variations were not significant.

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Fragmented CP1563 Shows Dose-dependent Anti-dyslipidemic Effects in vivo

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Figure 10 shows the results of the experiments that were conducted to confirm the

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reproducibility and dose-dependence of the effects of fragmented CP1563. The mice

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were divided into four groups: a HF diet-fed control group, a 0.25% fragmented

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CP1563-containing diet-fed group (approximately 4.0 × 1011 cells/day), a 0.5%

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fragmented CP1563-containing diet-fed group (approximately 8.0 × 1011 cells/day) and

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a 1% fragmented CP1563-containing diet-fed group (approximately 1.5 × 1010

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cells/day). The plasma HDL-cholesterol concentration increased (p < 0.01) and the AI

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decreased (p < 0.01) in a dose-dependent manner in the mice fed fragmented CP1563.

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No significant differences were observed in the liver or visceral fat weights after

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normalization to the body weight (Table 6).

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Correlations between Degree of CP1563 Fragmentation and PPARα Ligand Activity

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in vitro

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Figure 8 shows the PPARα agonist activity of the preparations of fragmented CP1563

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obtained using various fragmentation methods as described above. A significant

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negative correlation was detected between the diameters of the bacterial cells and the

322

PPARα activation ability (R2 = 0.3974, p < 0.01).

323

Correlations between Degree of CP1563 Fragmentation, PPARα Ligand Activity in

324

Vitro and HDL-cholesterol Level-Raising Activity in vivo

325

We found a clear negative correlation between the long dimension of CP1563

326

preparations and PPARα agonistic activities in vitro. We also found a correlation

327

between the PPARα ligand activities in vitro and the HDL-cholesterol-raising activity in

328

vivo (Table 7).

329

Discussion

330

In this study, we identified LAB strains whose diethyl ether extracts possessed PPARα/γ

331

agonist activity and found that fragmented cells of L. amylovorus CP1563 exerted a

332

recognizable anti-dyslipidemic effect in vivo. As shown by the luciferase reporter assays,

333

the highest PPARα activation was observed with the diethyl ether extract of CP1563,

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334

which also showed high PPARγ agonist activity (Fig. 1). High PPARα agonist activity

335

was detected with five of the seven tested L. amylovorus strains. According to our

336

screening test, L. amylovorus species may represent important PPARα agonists (Fig. 2).

337

CP1563 also showed higher PPARα agonist activity compared with the activities of

338

well-known food-related PPARα ligands (Fig. 3), even though their molecular

339

concentrations were higher than that of the CP1563 extract. These results suggest that

340

CP1563 serves as a possible dual agonist for PPARα/γ and could be included in food

341

sources for specific health uses. Based on these results, we selected CP1563 as a potent

342

dual agonist for subsequent animal experiments to investigate the anti-dyslipidemic

343

effects of CP1563 in vivo.

344

For the animal experiments, CP1563 cells were fragmented and mixed with food

345

to identify the PPAR-activating factor(s) derived from LAB cells. We hypothesize that

346

the component that originated from the fragmented bacterial cells was efficiently bound

347

by PPARα ligands in vivo. Previous studies revealed that the use of dead bacterial cells

348

or cell components could influence the host.23,24 We hypothesize that the activity of the

349

extracts was contained in TG-form fatty acids within the LAB cell membrane and that

350

the host’s pancreatic lipase may play an important role in the removal of fatty acid(s)

351

and the anti-dyslipidemic action of fragmented CP1563. However, fragmented CP1563

352

cells also activated PPARα in the CV1 cell culture system. This finding may suggest

353

that the active component(s) with the exception of those in TG form are released from

354

CP1563 cells by fragmentation. The fragmentation process is easier and more

355

cost-effective than the organic extraction process used by the food industry. In the same

356

model, mice fed a high-fat diet containing diethyl-ether extract from CP1563 at a level

357

equivalent to 1% fragmented CP1563 bacterial cells showed improvements in their lipid

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metabolism, but the effectiveness observed in these mice is rather meager compared

359

with that observed in the fragmented CP1563 preparation-fed mice (unpublished data).

360

These phenomena may have been observed due to differences in the digestibility in the

361

gastrointestinal tract. The organic solvent extract was directly exposed to the digestive

362

system, but the fragmented bacterial cell preparation may protect against a PPAR

363

activator(s). Thus, we subjected the strain to a practical processing method involving

364

rupturing of the bacterial bodies using DYNO-MILL.

365

We then examined the effects of possible PPAR ligands in CP1563 ether extracts

366

on the lipid metabolism of diet-induced obese mice. In our preliminary experiments, a

367

PPARα ligand-like effect was observed, as demonstrated by changes in plasma marker

368

molecules in the fragmented CP1563-fed group after six weeks of feeding (Fig. 5). In

369

contrast, no significant difference was observed after two weeks of feeding in the mice

370

fed the same concentration of CP1563 (Fig. 4). These results indicate that a minimum of

371

six weeks is needed to evaluate the anti-dyslipidemic effect of fragmented CP1563. An

372

increase in liver weight was also observed after six weeks of treatment (Table 3), and

373

this variable may serve as an indicator of PPARα activation caused by the high

374

expression of peroxisomes in the rodent liver. An increase in peroxisome by PPARα

375

activators in the liver reflects an increase in liver weight. This result suggests that the

376

observed increase in plasma HDL-cholesterol after six weeks of feeding may be related

377

to the activation of PPARα, which regulates the expression of ApoAI and ApoAII, genes

378

encoding the HDL-components apolipoprotein AI and AII, respectively. Thus, it is

379

possible that fragmented CP1563, similarly to fenofibrate, works as a PPARα activator.

380

As shown in Figure 7, PPARα activation and dyslipidemic effects were not

381

observed in the intact CP1563-fed mice. This result suggests that CP1563 exerts an

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382

anti-dyslipidemic effect more efficiently after fragmentation. We also found a negative

383

correlation between the length of the bacterial cells and PPARα activation (Fig. 8). A

384

smaller fragmentation size was correlated with higher PPARα activity and appears to

385

expose more active components from the cell preparations in both in vitro and in vivo

386

experiments. In this study, we fragmented CP1563 with a DYNO-MILL to obtain cells

387

lengths equal to approximately 68% of the original length. The results clearly indicate

388

that a component(s) of the bacterial cell is involved in the activation of PPARα and that

389

the fragmentation process is important for the anti-dyslipidemic effect of bacterial cells.

390

We hypothesize that the PPAR ligands expressed by CP1563 are found in the cell

391

membrane or cytoplasm and that these ligands are exposed on the surface upon

392

fragmentation, increasing the anti-dyslipidemic effect. These molecules may improve

393

the host’s lipid metabolism through the activation of PPARs.

394

The PPARα ligand-like effects of fragmented CP1563 were validated by

395

long-term administration and dose-dependence experiments. The effect of CP1563 may

396

be improved by the long-term administration or ingestion of high CP1563

397

concentrations. After 12 weeks of feeding, significant decreases were observed in the

398

plasma LDL-cholesterol and plasma TG levels, and an increase in HDL-cholesterol was

399

detected (Fig. 9). The TG levels did not change in the preliminary experiment, and the

400

changes in the TG concentrations were relatively slow compared with the changes in

401

HDL-cholesterol. The levels of high-molecular-weight adiponectin showed a tendency

402

to increase. It has been demonstrated that fibrates elevate the adiponectin level through

403

PPARα activation,31 and it is possible that the same pathway may be activated by

404

fragmented CP1563 in our experiments. These results suggest that an anti-dyslipidemic

405

effect was induced by CP1563 feeding. Although a decrease in visceral fat caused by

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PPARα activation was expected, no significant decrease was observed (Table 5). Figure

407

10 shows the dose-dependent increase in plasma HDL-cholesterol and decrease in AI

408

induced by fragmented CP1563 feeding and the reproducibility of the results. No

409

significant difference was noted with the combination of the dosage levels and the

410

feeding period (Table 6), and experiments at higher dosages will be conducted to

411

evaluate possible reductions in body fat. Paired feeding was employed in the series of

412

animal experiments, and it may be inappropriate to investigate the effect of the

413

fragmented preparations on the observed reduction in body fat. Recent studies have

414

demonstrated that the intestinal lipid metabolism, as regulated by PPARα, is a novel

415

target for decreasing the circulating lipid levels.32,33 However, a cholesterol-lowering

416

food may exert its effect by one or a combination of several mechanisms.16 Further

417

studies are needed to elucidate the mechanism underlying the anti-dyslipidemic action

418

of fragmented CP1563 and its extract in vivo.

419

In conclusion, we determined that a component(s) of CP1563 shows potent

420

PPARα/γ dual agonist activity and appears to improve dyslipidemia. An

421

anti-dyslipidemic effect was observed in diet-induced obese mice fed fragmented

422

CP1563, and the fragmentation process was found to be critical for activating PPARα

423

and exerting dyslipidemic effects in vivo. Further studies are needed to elucidate the

424

exact mechanism underlying the effects of fragmented CP1563 in vivo and to isolate the

425

possible active molecule(s).

426

Acknowledgments

427

We thank Dr. Izumi Yoshida and Dr. Yukiko Aoyama of Tempstaff Co., Ltd., for their

428

significant contribution to the preparation and editing of the manuscript.

429

Funding Sources

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This study was designed and funded by Asahi Group Holdings, Ltd. Futoshi Nakamura,

431

Yu Ishida, Daisuke Sawada, Nobuhisa Ashida, Tomonori Sugawara and Shigeru

432

Fujiwara are employees of Asahi Group Holdings, Ltd.

433

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References

435

1.

436 437

Kaur, J. A comprehensive review on metabolic syndrome. Cardiol. Res. Pract. 2014, 2014, 943162.

2.

Mulè, G.; Calcaterra, I.; Nardi, E.; Cerasola, G.; Cottone, S. Metabolic syndrome

438

in hypertensive patients: an unholy alliance. World J. Cardiol. 2014, 6(9),

439

890-907.

440

3.

441 442

Grundy, S. M. Small LDL, atherogenic dyslipidemia, and the metabolic syndrome. Circulation 1997, 95(1), 1-4.

4.

Bosomworth, N. J. Approach to identifying and managing atherogenic

443

dyslipidemia: a metabolic consequence of obesity and diabetes. Can. Fam.

444

Physician Medecin Famille Can. 2013, 59, 1169-1180.

445

5.

446 447

Mann, G. V. A factor in yogurt which lowers cholesteremia in man. Atherosclerosis 1977, 26(3), 335-340.

6.

Agerbaek, M.; Gerdes, L. U.; Richelsen, B. Hypocholesterolaemic effect of a new

448

fermented milk product in healthy middle-aged men. Eur. J. Clin. Nutr. 1995, 49,

449

346-352.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

450

7.

Bertolami, M. C.; Faludi, A. A.; Batlouni, M. Evaluation of the effects of a new

451

fermented milk product (Gaio) on primary hypercholesterolemia. Eur. J. Clin.

452

Nutr. 1999, 53(2), 97-101.

453

8.

Kawase, M.; Hashimoto, H.; Hosoda, M.; Morita, H.; Hosono, A. Effect of

454

administration of fermented milk containing whey protein concentrate to rats and

455

healthy men on serum lipids and blood pressure. J. Dairy Sci. 2000, 83(2),

456

255-263.

457

9.

Wang, C.; Wu, S.; Fang, J.; Wang, Y.; Shyu, Y. Cardiovascular and intestinal

458

protection of cereal pastes fermented with lactic acid bacteria in hyperlipidemic

459

hamsters. Food Res. Int. 2012, 48(2), 428-434.

460

10.

Ogawa, A.; Kadooka, Y.; Kato, K.; Shirouchi, B.; Sato, M. Lactobacillus gasseri

461

SBT2055 reduces postprandial and fasting serum non-esterified fatty acid levels

462

in Japanese hypertriacylglycerolemic subjects. Lipids Health Dis. 2014, 13(1),

463

36.

464

11.

Thompson, L. U.; Jenkins, D. J.; Amer, M. A.; Reichert, R.; Jenkins, A.;

465

Kamulsky, J. The effect of fermented and unfermented milks on serum

466

cholesterol. Am. J. Clin. Nutr. 1982, 36, 1106-1111.

ACS Paragon Plus Environment

Page 22 of 51

Page 23 of 51

Journal of Agricultural and Food Chemistry

467

12.

468 469

Massey, L. K. Effect of changing milk and yogurt consumption on human nutrient intake and serum lipoproteins. J. Dairy Sci. 1984, 67(2), 255-262.

13.

McNamara, D.; Lowell, A.; Sabb, J. Effect of yogurt intake on plasma lipid and

470

lipoprotein levels in normolipidemic males. Atherosclerosis 1989, 79(2–3),

471

167-171.

472

14.

Richelsen, B.; Kristensen, K.; Pedersen, S. B. Long-term (6 months) effect of a

473

new fermented milk product on the level of plasma lipoproteins--a

474

placebo-controlled and double blind study. Eur. J. Clin. Nutr. 1996, 50, 811-815.

475

15.

de Roos, N. M.; Schouten, G.; Katan, M. B. Yoghurt enriched with Lactobacillus

476

acidophilus does not lower blood lipids in healthy men and women with normal to

477

borderline high serum cholesterol levels. Eur. J. Clin. Nutr. 1999, 53(4), 277-280.

478

16.

479 480

cholesterol-lowering functional foods. J. Funct. Foods 2011, 3(2), 61-69. 17.

481 482

Chen, Z.; Ma, K. Y.; Liang, Y.; Peng, C.; Zuo, Y. Role and classification of

Grygiel-Górniak, B. Peroxisome proliferator-activated receptors and their ligands: nutritional and clinical implications--a review. Nutr. J. 2014, 13(1), 17.

18.

Lefebvre, P.; Chinetti, G.; Fruchart, J. C.; Staels, B. Sorting out the roles of PPAR

483

alpha in energy metabolism and vascular homeostasis. J. Clin. Invest. 2006,

484

116(3), 571-580.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

485

19.

486 487

Semple, R. K.; Chatterjee, V. K.; O'Rahilly, S. PPAR gamma and human metabolic disease. J. Clin. Invest. 2006, 116(3), 581-589.

20.

Munigoti, S.; Harinarayan, C. Role of Glitazars in atherogenic dyslipidemia and

488

diabetes: two birds with one stone? Indian J. Endocrinol. Metab. 2014, 18(3),

489

283-287.

490

21.

491 492

hypertension. World J. Cardiol. 2014, 6(8), 744-754. 22.

493 494

Usuda, D.; Kanda, T. Peroxisome proliferator-activated receptors for

Agrawal, R. The first approved agent in the Glitazar's class: saroglitazar. Curr. Drug Targets 2014, 15(2), 151-155.23.

23.

Kim, Y. I.; Hirai, S.; Goto, T.; Ohyane, C.; Takahashi, H.; Tsugane, T.; Konishi,

495

C.; Fujii, T.; Inai, S.; Iijima, Y.; Aoki, K.; Shibata, D.; Takahashi, N.; Kawada, T.

496

Potent PPARalpha activator derived from tomato juice,

497

13-oxo-9,11-octadecadienoic acid, decreases plasma and hepatic triglyceride in

498

obese diabetic mice. PLOS ONE 2012, 7, e31317.

499 500

24.

Adams, C. A. The probiotic paradox: live and dead cells are biological response modifiers. Nutr. Res. Rev. 2010, 23(01), 37-46.

ACS Paragon Plus Environment

Page 24 of 51

Page 25 of 51

Journal of Agricultural and Food Chemistry

501

25.

Taverniti, V.; Guglielmetti, S. The immunomodulatory properties of probiotic

502

microorganisms beyond their viability (ghost probiotics: proposal of

503

paraprobiotic concept). Genes Nutr. 2011, 6(3), 261-274.

504

26.

Takahashi, N.; Kawada, T.; Goto, T.; Yamamoto, T.; Taimatsu, A.; Matsui, N.;

505

Kimura, K.; Saito, M.; Hosokawa, M.; Miyashita, K.; Fushiki, T. Dual action of

506

isoprenols from herbal medicines on both PPARgamma and PPARalpha in

507

3T3-L1 adipocytes and HepG2 hepatocytes. FEBS Lett. 2002, 514(2–3), 315-322.

508

27.

Takahashi, N.; Kawada, T.; Goto, T.; Kim, C.; Taimatsu, A.; Egawa, K.;

509

Yamamoto, T.; Jisaka, M.; Nishimura, K.; Yokota, K.; Yu, R.; Fushiki, T. Abietic

510

acid activates peroxisome proliferator-activated receptor-gamma (PPARgamma)

511

in RAW264.7 macrophages and 3T3-L1 adipocytes to regulate gene expression

512

involved in inflammation and lipid metabolism. FEBS Lett. 2003, 550(1–3),

513

190-194.

514

28.

Miura, Y.; Hosono, M.; Oyamada, C.; Odai, H.; Oikawa, S.; Kondo, K. Dietary

515

isohumulones, the bitter components of beer, raise plasma HDL-cholesterol levels

516

and reduce liver cholesterol and triacylglycerol contents similar to PPARalpha

517

activations in C57BL/6 mice. Br. J. Nutr. 2005, 93(04), 559-567.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

518

29.

Shimura, M.; Hasumi, A.; Minato, T.; Hosono, M.; Miura, Y.; Mizutani, S.;

519

Kondo, K.; Oikawa, S.; Yoshida, A. Isohumulones modulate blood lipid status

520

through the activation of PPAR alpha. Biochim. Biophys. Acta. 2005, 1736,

521

51-60.

522

30.

Friedewald, W. T.; Levy, R. I.; Fredrickson, D. S. Estimation of the concentration

523

of low-density lipoprotein cholesterol in plasma, without use of the preparative

524

ultracentrifuge. Clin. Chem. 1972, 18, 499-502.

525

31.

Hiuge, A.; Tenenbaum, A.; Maeda, N.; Benderly, M.; Kumada, M.; Fisman, E.

526

Z.; Tanne, D.; Matas, Z.; Hibuse, T.; Fujita, K.; Nishizawa, H.; Adler, Y.; Motro,

527

M.; Kihara, S.; Shimomura, I.; Behar, S.; Funahashi, T. Effects of peroxisome

528

proliferator-activated receptor ligands, bezafibrate and fenofibrate, on

529

adiponectin level. Arterioscler. Thromb. Vasc. Biol. 2007, 27, 635-641.

530

32.

Uchida, A.; Slipchenko, M. N.; Cheng, J.; Buhman, K. K. Fenofibrate, a

531

peroxisome proliferator-activated receptor alpha agonist, alters triglyceride

532

metabolism in enterocytes of mice. Biochim. Biophys. Acta. 2011, 1811(3),

533

170-176.

534 535

33.

Kimura, R.; Takahashi, N.; Murota, K.; Yamada, Y.; Niiya, S.; Kanzaki, N.; Murakami, Y.; Moriyama, T.; Goto, T.; Kawada, T. Activation of peroxisome

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proliferator-activated receptor-alpha (PPARalpha) suppresses postprandial

537

lipidemia through fatty acid oxidation in enterocytes. Biochem. Biophys. Res.

538

Commun. 2011, 410(1), 1-6.

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Figure 1. Microbe PPAR activation assays. Relative activities were calculated as fold-changes compared with the positive controls, namely GW7647 (final concentration: 10 nM) for PPARα and troglitazone (final concentration: 1 µM) for PPARγ. (a) Relative PPARα-induction activities obtained with 46 strains of food microbes. (b) Relative PPARγ-activation activities of eight selected strains.

Figure 2. Results of a PPARα activation assay with L. amylovorus species. The relative luciferase activities of the seven L. amylovorus strains were calculated as fold-changes in PPARα induction compared with the activity of the positive control GW7647 (final concentration: 10 nM).

Figure 3. Comparison of the PPARα-induction activities of CP1563 and food-oriented PPARα ligands. Relative activities were calculated as fold-changes in PPARα induction compared with the activity of the positive control GW7647 (final concentration in the assay system: 10 nM). Epigallocatechin gallate (EGCG), eicosapentaenoic acid (EPA) and conjugated linoleic acid (CLA) were used as known food-oriented PPARα ligands at various concentrations.

Figure 4. Biological markers in the plasma of mice fed for two weeks. The HDL (A) and LDL cholesterol (B) and triglyceride (C) levels in 1% CP1563-fed mice and control diet-fed mice were measured, and the AI (D) was calculated based on these results. The values are expressed as the means ± S.D. (n = 12).

Figure 5. Biological markers in the plasma of mice fed for six weeks. The HDL (A)

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and LDL cholesterol (B) and triglyceride (C) levels in 1% CP1563-fed mice and control diet-fed mice were measured, and the AI (D) was calculated based on these results. The values are expressed as the means ± S.D. (n = 24). *p < 0.05 and ***p < 0.001 versus the control.

Figure 6. Photomicrographs of CP1563. (A) Unprocessed CP1563. (B) Fragmented CP1563.

Figure 7. Biological markers in the mouse plasma of mice fed unprocessed CP1563 and fragmented CP1563. The HDL (A) and LDL cholesterol (B) and triglyceride (C) levels in 1% CP1563-fed mice and control diet-fed mice were measured, and the AI (D) was calculated based on these results. The values are expressed as the means ± S.D. (n = 24). *p < 0.05 and ***p < 0.001 versus the control.

Figure 8. Correlation between the length of the fragmented bacterial cells and PPARα activation abilities in vitro. The relative PPARα activation abilities of fragmented CP1563 preparations obtained using nine different mills are presented as fold-changes compared with that of the positive control GW7647 (final concentration in the assay system: 10 nM). The final concentration of all of the fragmented CP1563 preparations was 625 µg/mL. The length of each fragmented CP1563 bacterium was measured using a scanning electron microscope.

Figure 9. Biological markers in the plasma of mice fed for 12 weeks. The HDL-cholesterol level (A), LDL-cholesterol level (B), triglyceride level (C), AI (D) and

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high-molecular-weight adiponectin level (E) in 1% CP1563-fed mice and control diet-fed mice were measured, and the values are presented as the means ± S.D. (n = 45). **p < 0.01 and ***p < 0.001 versus the control.

Figure 10. Dose-dependence. Linear regression between the concentrations of fragmented CP1563 in the experimental diets given to mice and the following indices: biological markers in mouse plasma, liver weight and visceral fat. The values shown are the means ± S.D. (n = 36). The R2 value indicates the coefficient of determination, and p indicates the probability of significance. (A) HDL cholesterol: R2 = 0.1003, p < 0.01. (B) LDL cholesterol: R2 = 0.3289, p < 0.05. (C) Triglycerides: R2 = 0.0046, p > 0.1. (D) AI: R2 = 0.0957, p < 0.01.

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Table 1. List of bacteria used for the PPAR assay and culture conditions Culture Plate temp. Species Strain agar (ºC)

Liquid medium

Bifidobacterium adolescentis

C211

37

BL

GAM

Bifidobacterium bifidum

C105

37

BL

GAM

Bifidobacterium breve

C21

37

BL

GAM

Bifidobacterium catenulatum

C1010

37

BL

GAM

Bifidobacterium infantis

C97

37

BL

GAM

Bifidobacterium longum

C76

37

BL

GAM

Enterococcus faecalis

C68

37

MRS

MRS

Enterococcus mundtii

C12

37

MRS

MRS

Issatchenkia orientalis

C04

37

MRS

MRS

Lactobacillus acidophilus

C611

37

MRS

MRS

37

MRS

MRS

CP1563, C48, C68, C611, Lactobacillus amylovorus C615, C85, ATCC33620 Lactobacillus brevis

C87

37

MRS

MRS

Lactobacillus buchneri

C98

37

MRS

MRS

Lactobacillus coryniformis

C814

37

MRS

MRS

Lactobacillus crispatus

C67

37

MRS

MRS

Lactobacillus delbrueckii

C51

37

MRS

MRS

C110, C25 L, C33

37

MRS

MRS

C23, C31, C32, C36

37

MRS

MRS

Lactobacillus delbrueckii subsp. bulgaricus Lactobacillus fermentum

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Lactobacillus gallinarum

C57

37

MRS

MRS

Lactobacillus gasseri

CP2305

37

MRS

MRS

Lactobacillus helveticus

C24, C26, C1010

37

MRS

MRS

Lactobacillus homohiochii

C99

37

MRS

MRS

Lactobacillus johnsonii

C63

37

MRS

MRS

Lactobacillus kefiri

C14, C15, C34S

37

MRS

MRS

Lactobacillus paracasei

C614

37

MRS

MRS

Lactobacillus parakefir

C83

37

MRS

MRS

Lactobacillus plantarum

C38, C810

37

MRS

MRS

Lactobacillus salivarius

C617

37

MRS

MRS

Lactococcus lactis

C512, C17, C510

30

MRS

MRS

Leuconostoc mesenteroides

C17

37

MRS

MRS

Saccharomyces cerevisiae

C27, C33 C13, C19

37

PD

PD

Saccharomyces unisporus

C13, C19

30

PD

PD

The strains highlighted in boldface type were used in both the PPARα and PPARγ assays. BL: glucose blood liver agar, MRS: de Man, Rogosa, and Sharpe medium, GAM: Gifu anaerobic medium agar. PD: potato dextrose.

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Table 2. Compositions of the experimental diets High-fat Fenofibrate

CP1563

Composition (%)

control

0.05%

0.50% 0.25% 0.50% 1.00%

Butter

15

15

15

15

15

15

Sucrose

52.45

52.45

52.45

52.45

52.45

52.45

Casein

20

20

20

20

20

20

Corn oil

1

1

1

1

1

1

Cellulose

5

4.95

4.5

4.75

4.5

4

AIN-93G mineral mixture

3.5

3.5

3.5

3.5

3.5

3.5

AIN-93 vitamin mixture

1

1

1

1

1

1

Choline chloride

0.25

0.25

0.25

0.25

0.25

0.25

Cysteine

0.3

0.3

0.3

0.3

0.3

0.3

Cholesterol

1

1

1

1

1

1

Sodium cholate

0.5

0.5

0.5

0.5

0.5

0.5

Fenofibrate

-

0.05

0.5

-

-

-

L. amylovorus CP1563

-

-

-

0.25

0.5

1

Total

100

100

100

100

100

100

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Table 3. Liver weights and visceral fat per body weight of mice. These values were obtained from preliminary experiments (A) Two weeks of feeding Body weight (g) Liver (mg/g)

Fat (mg/g) Initial

Final

n

Average

S.D.

Average

S.D.

Average

S.D.

Average

S.D.

Control

24

54.17

3.02

17.51

2.30

19.30

0.15

20.70

0.24

CP1563

24

53.08

5.18

17.00

2.08

19.31

0.18

19.50

0.24

Fenofibrate

24

66.13#

3.34

15.86

1.76

19.24

0.14

20.00

0.20

(B) Six weeks of feeding Body weight (g) Liver (mg/g)

Fat (mg/g) Initial

Final

n

Average

S.D.

Average

S.D.

Average

S.D.

Average

S.D.

Control

24

99.95

12.57

48.65

11.07

19.40

0.76

23.70

1.34

CP1563

24

110.68*

13.29

46.25

10.18

19.40

0.77

23.20

2.34

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Journal of Agricultural and Food Chemistry

Fenofibrate

24

122.42#

6.79

45.29

8.56

19.40

0.59

*p < 0.01 and #p < 0.001 versus the control.

ACS Paragon Plus Environment

24.30

0.97

Journal of Agricultural and Food Chemistry

Page 36 of 51

Table 4. Liver weights and visceral fat per body weight of mice fed fragmented and intact CP1563 Body weight (g) Liver (mg/g)

Fat (mg/g) Initial

n

Average S.D.

Final

Average S.D.

Average S.D. Average S.D.

30 61.66

3.85

41.36

11.40

18.42

0.46 22.22

1.26

Fragmented 30 63.52

6.12

46.14

11.47

18.44

0.59 22.97

1.18

30 63.03

3.49

43.53

12.22

18.41

0.57 22.56

1.47

Control

Intact

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Journal of Agricultural and Food Chemistry

Table 5. Liver weights, visceral fat per body weight, and changes in body weight of mice after 12 weeks of feeding Body weight (g) Liver (mg/g)

Fat (mg/g) Initial

n

Final

Average

S.D.

Average

S.D.

Average

S.D.

Average

S.D.

Control 50

57.34

3.35

82.79

15.71

18.36

0.71

29.43

2.16

CP1563 50

58.77

2.82

81.87

14.90

18.38

0.69

29.59

1.91

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Journal of Agricultural and Food Chemistry

Page 38 of 51

Table 6. Liver weights, visceral fat per body weight, and body weight changes of mice from the dose-dependence experiment Body weight (g) Liver (mg/g)

Fat (mg/g) Initial

n

Average S.D.

Final

Average

S.D.

Average

SD

Average SD

Control 36 60.51

2.43

49.76

10.37

18.72

0.56

23.17

1.19

0.25%

33 60.75

3.22

48.27

14.38

18.71

0.53

22.84

1.64

0.50%

32 61.26

2.42

52.50

11.19

18.71

0.52

23.44

1.31

1.00%

35 60.68

2.80

52.21

12.91

18.72

0.58

23.08

1.56

539

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Journal of Agricultural and Food Chemistry

Table 7. Correlations between degree of CP1563 fragmentation, PPARα ligand activity in vitro and HDL-cholesterol level-raising activity in vivo

Intact CP1563

Mildly

Moderately

Highly

fragmented

fragmented

fragmented

(by a jet-mill) Average

S.D.

Bacterial long

(by a DYNO-MILL)

(by a planetary Mill)

Average

S.D.

Average

S.D.

Average

S.D.

2.405

1.114

1.892

1.032

0.455

0.5065

2.773 dimensiona

1.079

(n=1003)

(n=731)

(n=722)

p